Cranial and trunk neural crest cells use different ... - Caltech Authors

Development 116, 531-541 (1992) Printed in Great Britain © The Company of Biologists Limited 1992

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Cranial and trunk neural crest cells use different mechanisms for attachment to extracellular matrices THOMAS LALLIER 1,*, GABRIELLE LEBLANC2, KRISTIN B. ARTINGER1 and MARIANNE BRONNERFRASER1,† 1Developmental 2Department *Present †Author

Biology Center, University of California, Irvine, CA 92717, USA of Anatomy, School of Dentistry, Oregon Health Sciences University, Portland, OR 97201, USA

Address: University of Virginia, Health Science Center, Box 439, Department of Anatomy and Cell Biology, Charlottesville, VA 22908, USA for correspondence

Summary We have used a quantitative cell attachment assay to compare the interactions of cranial and trunk neural crest cells with the extracellular matrix (ECM) molecules fibronectin, laminin and collagen types I and IV. Antibodies to the 1 subunit of integrin inhibited attachment under all conditions tested, suggesting that integrins mediate neural crest cell interactions with these ECM molecules. The HNK-1 antibody against a surface carbohydrate epitope under certain conditions inhibited both cranial and trunk neural crest cell attachment to laminin, but not to fibronectin. An antiserum to 1 integrin inhibited attachment of trunk, but not cranial, neural crest cells to laminin and collagen type I, though interactions with fibronectin or collagen type IV were unaffected. The surface properties of trunk and cranial neural crest cells differed in several ways. First, trunk neural crest cells attached to collagen types I and IV, but cranial neural crest cells did not. Second, their divalent cation requirements for attachment to ECM molecules differed. For fibronectin substrata, trunk neural crest

cells required divalent cations for attachment, whereas cranial neural crest cells bound in the absence of divalent cations. However, cranial neural crest cells lost this cation-independent attachment after a few days of culture. For laminin substrata, trunk cells used two integrins, one divalent cation-dependent and the other divalent cation-independent (Lallier, T. E. and BronnerFraser, M. (1991) Development 113, 1069-1081). In contrast, cranial neural crest cells attached to laminin using a single, divalent cation-dependent receptor system. Immunoprecipitations and immunoblots of surface labelled neural crest cells with HNK-1, 1 integrin and 1 integrin antibodies suggest that cranial and trunk neural crest cells possess biochemically distinct integrins. Our results demonstrate that cranial and trunk cells differ in their mechanisms of adhesion to selected ECM components, suggesting that they are non-overlapping populations of cells with regard to their adhesive properties.

Introduction

underneath the ectoderm, and produce sensory and sympathetic ganglia, adrenal chromaffin cells and melanocytes. Sacral neural crest cells (arising below the level of somite 28) give rise to enteric ganglia and to some sympathetic and sensory ganglion cells (Pomeranz and Gershon, 1990; Serbedzija et al., 1991). The differing developmental fates of different rostrocaudal populations of neural crest cells could reflect (1) intrinsic differences in their developmental potentials and/or (2) differences in the environmental cues that they encounter after their migration from the neural tube. The developmental potentials of cranial and trunk neural crest cells have been compared using both in vivo transplantation and in vitro techniques. These experiments have shown that cranial and trunk neural crest cells share the ability to produce melanocytes, glia, sensory neurons and various kinds of autonomic neurons (Niu, 1947; Le Douarin and Teillet,

The neural crest is a migratory cell population that arises from the dorsal portion of the neural tube in vertebrate embryos. These cells migrate extensively and give rise to a large range of derivatives. Along the neural axis, there are four main subdivisions of the neural crest: cranial, vagal, trunk and sacral (LeDouarin, 1982). These different rostrocaudal populations follow different migratory pathways and produce different sets of derivatives. Cranial neural crest cells (arising from presomitic levels) migrate through the cranial mesenchyme and produce facial cartilage and bone, and cranial parasympathetic and sensory ganglia. Vagal neural crest cells (arising at the level of somites 1-7) invade the gut, migrate caudally and populate the enteric ganglia. Trunk neural crest cells (arising at the level of somites 8-28) migrate through the somites and

Key words: integrins, cell adhesion, laminin, fibronectin, collagen.

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T. Lallier and others

1974; Le Douarin et al., 1975; Kahn et al., 1980; Le Lievre et al., 1980; Fauquet et al., 1981; Leblanc et al., 1990). However, only cranial neural crest cells have the ability to produce cartilage and bone (Raven, 1931; Nakamura and Ayer-Le Lievre, 1982). In addition, some cranial neural crest cells produce fibronectin (a characteristic mesenchymal secretion product) when grown in vitro, whereas trunk neural crest cells do not (Newgreen and Thiery, 1980). Taken together, these experiments indicate that cranial and trunk neural crest cells share some properties, but also have some inherent differences, particularly in their ability to produce mesenchymal derivatives. Differences in migratory properties of cranial and trunk neural crest cells also have been noted (Le Douarin and Teillet, 1974; Le Lievre et al., 1980; Bronner-Fraser, 1985, 1986, 1987; Bronner-Fraser and Lallier, 1988). Neural crest cell migratory pathways contain many extracellular matrix (ECM) molecules including fibronectin (Newgreen and Thiery, 1980), laminin (Krotoski et al., 1986), tenascin/cytotactin (Tan et al., 1987), collagens (Duband and Thiery, 1987; Perris et al., 1991b) and proteoglycans (Erickson, 1988; Perris et al., 1991a) which are thought to influence their migration and differentiation. Perturbation experiments using antibodies that recognize ECM components or their receptors have demonstrated the importance of the ECM for proper migration of some neural crest cell populations. For example, antibodies to fibronectin (Poole and Thiery, 1986), tenascin (Bronner-Fraser, 1988) and a laminin-heparan sulfate proteoglycan complex (Bronner-Fraser and Lallier, 1988) can disrupt cranial neural crest cell migration in vivo. Cranial neural crest cell migration also is blocked by antibodies against the β1 subunit of integrin (Bronner-Fraser, 1985, 1986) or against the HNK-1 carbohydrate epitope (Bronner-Fraser, 1987). Neural crest cell interactions with ECM molecules may be mediated by a class of receptors called integrins (Hynes, 1987). An integrin receptor is a heterodimer consisting of two subunits, α and β, that are non-covalently linked. These transmembrane glycoproteins mediate cell adhesion to a variety of ECM molecules including fibronectin, laminin, vitronectin and various collagens (Horwitz et al., 1985; Buck et al., 1986; Hynes, 1987; Tomaselli et al., 1988). Integrin α subunits possess binding sites for divalent cations that are thought to be required for function (Cheresh et al., 1987; Edwards et al., 1988; Ignatius and Reichardt, 1988; Smith and Cheresh, 1988). Many integrins appear to have a preference for specific divalent cations. For instance, α2β1 integrins recognize collagens in a magnesium-dependent manner, while α3β1 integrins recognize collagens in a calcium-dependent manner. This cation selectivity may be a useful tool for classifying different integrins. Neural crest cells have several β1-integrins; for example, trunk neural crest cells have an α1β1 heterodimer which bears an HNK1 epitope (Lallier and Bronner-Fraser, 1992), as well as at least one other uncharacterized β1 heterodimer. Cranial neural crest cells also have immunocytochemically detectable β1-integrins (Krotoski et al., 1986), but their functional properties are not understood. One approach for elucidating possible differences between migrating cranial and trunk neural crest cells is to characterize their cell surface properties, such as the recep-

tors that mediate their interactions with the ECM. In the present study, we use a centrifugation assay (McClay et al., 1981) to analyze the adhesive interactions of neural crest cells with laminin, fibronectin, and collagen types I and IV. We compare the abilities of cranial and trunk neural crest cells to attach to these substrata. We utilize α1 integrin, β1 integrin and HNK-1 antibodies to inhibit neural crest cellsubstratum interaction under various conditions and to characterize their surface properties biochemically. The results suggest that cranial and trunk neural crest cells differ in the receptors that they use for attachment to these substrata.

Materials and methods Materials Extracellular matrix glycoproteins (mouse laminin, human fibronectin, rat collagen type I and bovine collagen type IV) were purchased from Collaborative Research Inc. (Bedford, MA). JG22 hybridoma cells were purchased from Developmental Studies Hybridoma Bank (University of Iowa). The HNK-1 antibody producing hybridoma cell line was purchased from ATCC (Rockville, MD). The anti-α1 integrin antiserum was the generous gift of Dr Mats Paulsson.

Cranial neural crest cell primary cultures Primary cranial neural crest cultures were prepared from the neural tubes of Japanese quail embryos (Coturnix coturnix japonica) as previously described (Leblanc et al., 1990). Briefly, embryos were incubated for 30-34 hours, at which time their developmental age was comparable to that of chick stages 8-9. Embryos were dissected in Howard Ringer’s solution using electrolytically sharpened tungsten needles. The neural folds were excised from the mesencephalic region, and the explants were placed in a few drops of culture medium on 35 mm plastic dishes coated with human fibronectin (25 µg/ml). After 1 hour, 1.5 ml of culture medium was added to the plates. The culture medium used was MEM (Grand Island Biological Co., GIBCO) supplemented with 15% horse serum (GIBCO) and 10% 11-day chick embryo extract, which was prepared as described previously (Howard and Bronner-Fraser, 1985). Within a few hours after explantation, numerous neural crest cells migrated from the explant to form a monolayer on the plate.

Trunk neural crest cell primary cultures Primary trunk neural crest cultures were prepared from the neural tubes of Japanese quail embryos (Coturnix coturnix japonica; Bronner-Fraser, 1985). Embryos were incubated for 48 hours, at which time their developmental age was comparable to that of chick stages 13-15 (Hamburger and Hamilton, 1950). The region of the trunk consisting of the six to nine most posterior somites as well as the unsegmented mesenchyme was dissected away from the embryo. The neural tubes were isolated by trituration in 160 units/ml of collagenase (Worthington Biochemical, Freehold, NJ). After stopping the enzymatic reaction with MEM containing 15% horse serum and 10% chick embryo extract, neural tubes were plated onto fibronectin-coated culture dishes. During the next several hours, the neural crest cells migrated away from the explant and onto the culture dish.

Cell preparation for attachment assays Neural crest cultures for use in quantitative attachment assays were labelled by the addition of [3H]leucine (10-50 µCi/ml) to their culture media 4 hours after explantation. The cells were

Integrins on cranial and trunk neural crest cells allowed to incorporate labelled leucine for 16 hours before use in the assay. Cultures were scraped to remove the neural tube, notochord and occasional somite cells. The remaining neural crest cells were rinsed five times with blocking MEM [bMEM; minimal essential media (Gibco) containing 0.5 mg/ml ovalbumin (Sigma)]. Cells were removed by incubation in 5 mM EDTA in bMEM for 10 minutes at 37°C. This procedure removed ~95-98% of the cranial neural crest cells and 98% of trunk neural crest cells.

Cell attachment assay Cell-substratum adhesion was measured as described by McClay et al. (1981) and modified for assaying neural crest cells (Lallier and Bronner-Fraser, 1991). Briefly, fibronectin, laminin or collagens were coated at the indicated concentrations in carbonate buffer (100 mM, pH 8.0; containing 1 mM CaCl2 and 500 µM MgCl2) onto polyvinyl chloride (PVC) microtitre plates by adsorption for 12 hours at 25°C. Protein substrata were plated at concentrations of 20 µg/ml (fibronectin and collagen type IV) and 100 µg/ml (collagen type I). LN-Ca2+ and LN-EDTA were prepared by coating plates with 20 µg/ml of laminin in a solution containing 5 mM Ca 2+ or EDTA, respectively. Wells were washed extensively with PBS (5 times) and incubated with bMEM for 2 hours at 25°C. Ovalbumin was routinely added to block nonspecific binding. Wells were rinsed and filled with either bMEM containing the antibody to be tested, or blocking phosphate buffer (bPBS; 10 mM phosphate buffer, pH 7.4 containing 150 mM NaCl and 0.5 mg/ml ovalbumin) containing the divalent cation concentration to be tested. Aliquots of [3H]leucine-labelled neural crest cells, rinsed to remove excess EDTA, were added to each test well. Individual wells were seeded with 500 to 1000 cells, which were deemed to be ~90% pure neural crest cells based on morphological criteria. In those cases where cell adhesion was measured in the absence of divalent cations, cells were resuspended in Ca2+/Mg2+-free PBS containing 0.5 mg/ml ovalbumin and 1.5 mM EDTA. Neural crest cells were brought into contact with the substratum molecules by a centrifugal force of 150 g for 5 minutes. Chambers were sealed and incubated for 15 minutes at 37°C. Neural crest cells that failed to adhere strongly to the substratum molecules were removed by a centrifugal force of 50 g for 5 minutes. The chambers were quickly frozen in a methanol/dry ice bath. The uncoated wells (unbound cells) and substratum-coated wells (bound cells) were removed and placed separately into scintillation vials. Samples were analyzed for 3H counts per minute (c.p.m.) using a scintillation counter (Beckman LS 5801) in order to determine the percentage of counts bound to the substratum. Six wells were counted per experiment and three experiments were compared for consistency. Within these experiments, non-specific binding to ovalbumin ranged from 5 to 15% of the counts present within a given well. Variability between experiments using identical conditions was ~10%. This variability is likely to be due to slight differences within the population of neural crest cells. Two values for percentage of neural crest cell attachment were determined as significantly different when the P value, by Student’s one-sided t-test, was 0.05), with approximately 65% of the cells binding under each of these conditions (Fig. 1). The addition of Mn2+, however, significantly inhibited cranial neural crest cell attachment to fibronectin (P0.05; Fig. 3). In contrast, trunk neural crest cell attachment to LN-Ca2+ did not require divalent cations, while attachment to LN-EDTA was divalent cation dependent with a preference for Ca2+ and Mn2+ over Mg2+ (Lallier and Bronner-Fraser, 1992). Collagens In parallel experiments, we tested the divalent cation dependence of cranial and trunk neural crest cell attachment to collagens (Fig. 4). We were unable to detect significant attachment of cranial neural crest cells to collagen types I or IV in either the presence or absence of divalent cations (P>0.05). Trunk neural crest cells attached to both collagens I and IV (approximately 45% and 35% bound to each substratum, respectively) in a divalent cation-dependent manner. The addition of either Ca2+ or Mn2+ resulted in comparable levels of attachment to collagen type I (approximately 25%; P0.05). In contrast, cranial neural crest cell attachment to both LN-EDTA and LN-Ca2+ was partially (about 50%) inhibited by the HNK1 antibody, but was unaffected by the α1 antiserum (P>0.05).

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Fig. 5. Antibody inhibition of cranial and trunk neural crest cell attachment to fibronectin substrata. Cell attachment was assayed in the presence of 50 µg/ml HNK-1 antibody, 50 µg/ml JG22 antibody or ~10 µg/ml of anti-α1 antiserum. Bars represent the mean of at least six wells and the error represent the s.e.m.

Fig. 7. Antibody inhibition of trunk neural crest cell attachment to collagen type I and IV substrata. Cell attachment was assayed in the presence of 50 µg/ml HNK-1 antibody, 50 µg/ml JG22 antibody or ~10 µg/ml of anti-α1 antiserum. Bars represent the mean of at least six wells and the error represent the s.e.m.

IV. The JG22 antibody abolished attachment of trunk neural crest cells to both collagen types I and IV at antibody concentrations of 50 µg/ml (Fig. 7; P0.05). The α1 antiserum completely blocked attachment to collagen type I, but had no effect on attachment to collagen type IV.

Fig. 6. Antibody inhibition of cranial and trunk neural crest cell attachment to laminin substrata. Laminin substrata were plated in the presence of (A) 5 mM Ca2+, or (B) 5 mM EDTA. Cell attachment was assayed in the presence of 50 µg/ml HNK-1 antibody, 50 µg/ml JG22 antibody or ~10 µg/ml of anti-α1 antiserum. Bars represent the mean of at least six wells and the error represent the s.e.m.

Collagens Trunk neural crest cells were tested for the effects of blocking antibodies on their attachment to collagen types I and

Identification of the HNK-1-positive protein on cranial neural crest cells Surface biotinylated cranial neural crest cells were immunoprecipitated using the HNK-1 antibody and then transferred to nitrocellulose membranes for detection using 125I-streptavidin (Fig. 8A). A major band at 180×103 M r and two minor bands of 165×103 Mr and 120×103 Mr were detected. In previous studies of trunk neural crest cells (Lallier and Bronner-Fraser, 1991, 1992), the major HNK-1 immunoreactive protein was determined to be a 165×103 Mr α1 integrin subunit. The 120×103 Mr probably corresponds to the chick β1 subunit of integrin. To examine further the relationship between the HNK-1 epitope and integrins, surface labelled cranial neural crest cells were immunoprecipitated with JG22 (anti-β1 integrin), anti-α1 integrin or HNK-1 antibodies. The immunoprecipitates then were immunoblotted with anti-α1 or HNK-1 antibodies (Fig. 8B). On the JG22 lane, the anti-α1 antiserum recognizes a 165×103 Mr and a 120×103 Mr band, whereas the HNK-1 antibody recognizes a 180×103 Mr band. Because the JG22 antibody precipitates both the β1 integrin subunit and associated α subunits, the 165×103 Mr band probably corresponds to the α1 subunit whereas the 180×103 Mr band corresponds to an unidentified α integrin subunit. The 120×103 Mr band corresponds to the β1 subunit, for which the antiserum has some reactivity. The HNK-1 antibody does not recognize any bands on anti-α1 antiserum immunprecipitates or vice versa. In contrast, in

Integrins on cranial and trunk neural crest cells

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Fig. 8. (A) Immunoprecipitation of cranial neural crest cells with the HNK1 antibody. Lane 1 represents control immunoprecipitates using BSA-coupled Sepharose 4B, while lane 2 represents the immunoprecipitation using the HNK-1 antibody coupled to Sepharose 4B. The HNK-1 antibody immunoprecipitates a major protein band of 180×103 Mr from surface labelled neural crest cells. In addition, two minor protein bands were detected, with Mr of 165×103 and 120×103. The lines correspond to relative molecular masses of 180, 165 and 120 ×103, respectively. (B) Immunoprecipitations of surface-labelled cranial neural crest cells with JG22 (anti-β1 integrin), anti-α1 integrin or HNK-1 antibodies followed by immunoblots with anti-α1 integrin antibodies (lanes 1 to 4) or HNK-1 antibodies (lanes 5 to 8). Lanes 1 and 5 = JG22 immunoprecipitate; lanes 2 and 6 = α1 immunoprecipitates; lanes 3 and 7 = HNK-1 immunoprecipitates; lanes 4 and 8 = control lanes with no primary antibody. The α1 antibody immunoblots a 165×103 Mr band in both the JG22 and α1 immunoprecipitates, corresponding to the α1 subunit; it also recognizes a 120×103 Mr band corresponding to the β1 subunit. The HNK-1 antibody immunoblots a 180×103 Mr band on the JG22 lane on and on itself; because this band co-precipitates with β1-integrin, it corresponds to an unidentified α integrin subunit. HNK-1 antibody does not recognize any bands on α1 antibody immunprecipitates or vice versa. The lines correspond to relative molecular masses of 180, 165 and 120 ×103, respectively.

immunoprecipitates of trunk neural crest cells, HNK-1 and anti-α1 integrin antibodies recognize the same 165×103 Mr band (Lallier and Bronner-Fraser, 1992). This biochemical evidence suggests that the predominant 180×103 Mr HNK1 epitope on cranial neural crest cells is an α integrin subunit other than α1, in agreement with the functional studies. Microinjection of anti- integrin antiserum onto cranial neural crest pathways in vivo Our biochemical data suggest that cranial neural crest cells possess α1 integrin subunits. However, in the cell adhesion assay, antibodies to this receptor do not block the attachment of cranial neural crest cells, suggesting it may be nonfunctional. We examined further the functional properties of this receptor by introducing α1 integrin antiserum onto cranial neural crest migratory pathways in situ. Previous studies have showed that antibodies against the β1 subunit of integrin interfere with the normal migration of cranial neural crest cells when introduced into the cranial mesenchyme of 3- to 9-somite-stage embryos (Bronner-Fraser, 1985, 1986). One day after microinjection of anti-α1 antiserum (n = 9 embryos), all embryos appeared morphologically normal. This contrasts with numerous other antibodies that block cell-matrix interactions, many of which perturb cranial neural crest migration (Bronner-Fraser, 1985, 1986; Bronner-Fraser, 1988; Bronner-Fraser and Lallier, 1988). These results are consistent with the in vitro data suggesting that cranial neural crest cells do not utilize the α1 integrin subunit for attachment to extracellular matrices. Discussion We have compared the adhesion of cranial and trunk neural crest cells to various ECM molecules with respect to both divalent cation dependence and antibody sensitivity. The

results are summarized in Table 1. Attachment of both cranial and trunk neural crest cells to all of the tested substrata was eliminated by anti-β1 integrin antibodies, indicating that their interactions are mediated by integrins. However, the characteristics of cell attachment to fibronectin, laminin and collagen were different for cranial versus trunk neural crest cells. To bind fibronectin, cranial neural crest cells used a divalent cation-independent integrin, whereas trunk neural crest cells used a divalent cation-dependent integrin. To attach to laminin, cranial neural crest cells used a divalent cation-dependent integrin that was sensitive to addition of HNK-1 antibody. In contrast, trunk neural crest cell

Table 1. Summary of results for neural crest cell attachment to laminin Antibody

Divalent cations

Substratum Cell type

HNK-1

JG22

EDTA

Fibronectin Cranial Trunk

= =

− −

+ −

+ +

+ +

− +

+ +

Laminin-EDTA Cranial Trunk

− =

− −

− −

+ +

+ −

+ +

+ +

Laminin-Ca2+ Cranial Trunk

− −

− −

− +

+ +

+ +

+ +

+ +

Collagen I Cranial Trunk

N.A. −

N.A. −

− −

− +

− −

− +

− +

Collagen IV Cranial Trunk

N.A. =

N.A. −

− −

− −

− −

− −

− +

+ promotes cell attachment = allows cell attachment − inhibits cell attachment N.A. not assayed

Ca2+ Mg2+ Mn2+ Ca/Mg

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attached to laminin via two different mechanisms, both distinct from that observed on cranial cells. In the case of collagens, cranial neural crest cells failed to bind to collagen types I or IV, whereas trunk neural crest cells bound to both collagens by divalent cation-dependent integrins. The results suggest that: (1) both trunk and cranial neural crest cells possess numerous integrins which can be distinguished by their cation dependency, sensitivity to antibodies and biochemical profiles; and (2) cranial and trunk neural crest cells are non-overlapping populations with respect to their cell-surface adhesive properties. The divalent cation dependence of cranial and trunk neural crest cell binding differs for each substratum tested. By comparing their cation requirements for different substrata with those of previously characterized integrins (summarized in Table 2), we might get some insight into the distinct types of integrins used by neural crest cells. Trunk and ‘old’ cranial neural crest cells attach to fibronectin by means of β1 integrins which use Ca2+, Mg 2+ or Mn 2+; this is consistent with the known properties of α2β1, αvβ1, or αvβ3 integrins (Kirchhofer et al., 1990; Dedhar and Gray, 1990; Vogel et al., 1990; Charo et al., 1990). In contrast, ‘young’ cranial neural crest cells attach to fibronectin through a divalent cation-independent integrin, with unique Table 2. Divalent cation dependence of integrin receptors Beta

Alpha

Calcium dependent 1 1 1 1 1 3 1 3 1 5 1 5 3 v 3 IIb

Ligand LM LM,Col 1-4 LM Col 1,4 FN FN VN,RGD FN,Fb,VN

Divalent cations utilized Ca2+ Mg2+ Mn2+ + + + + + + + 2+

+

+

+ + 2+

2+

Magnesium dependent 1 1 LM,Col 4 1 2 Col 1 2 Col 1,2,3,4 1 2 Col 1,2,3,4,6 1 3 LM,FN 1 6 LM 1 v FN,RGD 3 IIb FN,VN

? ? ? − − − −

+ 2+ 2+ 2+ + + + +

Divalent cation dependent 1 180k LM 1 130k LM

? ?

? ?

References 1 Belkin et al., 1990 2 Carter et al., 1990 3 Edwards et al., 1987 4 Edwards et al., 1988 5 Elices et al., 1991 6 Gailit and Ruoslahti, 1988 7 Ignatius and Reichardt, 1988 8 Kirchhofer et al., 1991 9 Kramer et al., 1989 10 Rossino et al., 1990 11 Sonnenberg et al., 1988 12 Staatz et al., 1989 13 Staatz et al., 1990 14 Turner et al., 1987 15 Wayner and Carter, 1987

+ +

+

Reference 7 1 2 5 3 6 8 6

+

14 15 12 13 5 11 8 4

? ?

10 9

2+ +

binding characteristics distinct from all previously reported integrins. Cranial neural crest cells bind to laminin via β1 integrin(s) that show no selectivity for divalent cations, and therefore may be α2β1 integrins (Sonnenberg et al., 1988, 1990; Hall et al., 1990; Shaw et al., 1990; Dedhar and Saulnier, 1990; Kramer et al., 1990; Shimizu et al., 1990) or α6β4 integrins (Elices and Hemler, 1989; Lotz et al., 1990; Lee et al., 1992). Trunk neural crest cells adhere to some laminin conformations via a divalent cation-independent integrin, while attaching to other laminin conformations through an integrin that utilizes Ca2+ or Mn2+, but not Mg2+. These divalent cation requirements differ from those of any known integrin. Trunk neural crest cells adhere best to both collagen type I and IV in the presence of both Ca2+ and Mg2+, consistent with the properties of α1β1 integrins (Turner et al., 1987; Belkin et al., 1990). In contrast, cranial neural crest cells do not attach to either collagen type. An interesting and surprising observation from our functional studies is that two of the integrins on neural crest cells are divalent cation-independent, in contrast to mostly previously characterized integrins which require divalent cations for function. One cation-independent integrin is on cranial neural crest cells and recognizes fibronectin; the other is on trunk neural crest cells and recognizes laminin (Lallier and Bronner-Fraser, 1991, 1992). These integrins appear to be temporally regulated since they are gradually lost with age. Because the majority of investigators use older cell populations or cell lines for studying cell-matrix adhesion, these divalent cation-independent integrins may have been missed previously. Thus, the mechanisms of cell attachment used by embryonic cells may be distinct from those employed by stable or metastatic cell types. The HNK-1 antibody, which recognizes a surface carbohydrate found on neural crest cells, inhibits the attachment of cranial neural crest cells to laminin and trunk neural crest cells to collagen type I and laminin. The inhibition is partial (~50%), probably reflecting the fact that not all neural crest cells express the HNK-1 epitope (Teillet et al., 1987; Maxwell and Forbes, 1988). For example, we found that approximately 25% of trunk neural crest cells and 50% of cranial neural crest cells lack detectable HNK-1 immunoreactivity after two days in vitro (unpublished observation). Previous studies have shown that injection of the HNK-1 antibody into the cranial mesenchyme during neural crest cell migration causes abnormal morphogenesis (BronnerFraser, 1987). Similarly, antibodies against the β1 subunit of integrin (Bronner-Fraser, 1985, 1986) or a lamininheparan sulfate proteoglycan complex (Bronner-Fraser and Lallier, 1988) inhibit migration of a subpopulation of cranial neural crest cells. These results are consistent with the idea that neural crest-laminin interactions may be required for their normal migration. Taken together, these studies suggest that the HNK-1 epitope appears to be important for the recognition of laminin by some cranial neural crest cells both in vivo and in vitro. Our data suggest that the proteins bearing the HNK-1 epitope are distinct for cranial versus trunk neural crest cell populations. First, the HNK-1-sensitive adhesion mechanism to laminin is divalent cation-independent for trunk neural crest cells, but divalent cation-dependent for cranial neural crest cells. Second, biochemical analysis suggests

Integrins on cranial and trunk neural crest cells that the HNK-1 antibody recognizes a 180×103 Mr glycoprotein, corresponding to an unidentified α integrin subunit, on cranial neural crest cells. In contrast, the epitope appears to be associated with a 165×103 Mr α1 integrin subunit on trunk neural crest cells (Lallier and Bronner-Fraser, 1991, 1992). Furthermore, immunoprecipitates of cranial neural crest cells using the chick α1 integrin antibody recognize a 165×103 Mr that is not recognized by the HNK-1 antibody in immunoblots, and vice versa. Thus, both biochemical and functional studies indicate that the HNK-1-bearing glycoprotein is different on cranial and trunk neural crest cells. Although both trunk and cranial neural crest cells possess a biochemically detectable α1 integrin subunit, the data demonstrate that the anti-α1 integrin antiserum effects trunk but not cranial neural crest cell attachment. Thus, cranial and trunk neural crest cells may express some identical integrins that differ in functional properties. Previous work suggests that the functional characteristics of a particular integrin heterodimer may differ depending upon the cell type in which it is expressed. For example, the α2β1 integrin on human endothelial cells recognizes laminin and collagens, while the same receptor heterodimer on platelets only recognizes collagens (Kirchhofer et al., 1990). This study demonstrates that cranial and trunk neural crest cells differ in (1) the range of ECM molecules to which they can adhere; (2) the divalent-cation requirements and antibody sensitivities of the integrins that mediate these interactions; and (3) the biochemical profile of their integrins. These results suggest that cranial and trunk neural crest cells are non-overlapping populations with respect to their cell surface adhesive properties. In situ, cranial and trunk neural crest cells give rise to different, but overlapping, sets of derivatives. Both cranial and trunk neural crest cells produce peripheral neurons and glia, but only cranial neural crest cells produce cartilage and bone. The commonalities in the developmental potentials of cranial and trunk neural crest cells are not reflected in their cell-surface adhesive properties. We observed no similarities between cranial and trunk neural crest cells in terms of their mechanisms of adhesion to specific ECM molecules. This result suggests that differences in cell-surface properties are not necessarily associated with differences in differentiative potential. Furthermore, in vivo transplantation experiments suggest that cranial and trunk neural crest cells share the capacity to translocate along heterologous neural crest migratory pathways (LeDouarin and Teillet, 1974; LeLievre et al., 1980) despite the fact that their surface properties differ. However, there are numerous differences between the migratory behavior of cranial and trunk neural crest cells. After transplantation to the trunk region, some of the cranial crest cells invade the gut and populate enteric ganglia and Remak’s ganglion, sites that would not normally be populated by trunk neural crest cells. In addition, antibody perturbation experiments indicate that cell-matrix interactions are required for the proper migration of cranial neural crest cells, whereas they may have a lesser role in the trunk (Bronner-Fraser, 1985, 1986, 1987; BronnerFraser and Lallier, 1988). It is possible that the differences that we have observed in the cell-surface properties of cranial and trunk neural crest cells contribute to these differences in their migratory behavior.

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In conclusion, our results, based on antibody sensitivity, divalent cation dependence and substratum specificity, indicate that cranial and trunk neural crest cells use different integrins to adhere to the extracellular matrix. The migratory pathways followed by these two cell populations during normal development in vivo are distinct, as are their sets of derivatives. The present results, together with these previous findings, sum to indicate that neural crest cells arising from different axial levels are distinct populations with respect to their cell-surface properties as well as their developmental potentials. This work was supported by USPHS grant HD-15527 to M. B. F. and BNS-8702512 to G. G. L.

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